The defining properties of the hematopoietic stem cell (HSC) are to undergo both differentiation and self renewal. Without the ability to self renew, an HSC would not be able to maintain the ability to replenish the short lived cells of the blood and lymph. However, the expansion of self-renewing cells must be strictly regulated or the cells will give rise to a malignancy. Therefore, understanding the mechanisms by which self renewal is regulated is crucial to understanding both normal development and cancer. In the past funding period, our group has shown that BMI1 is required for the maintenance of normal blood and brain stem cells, and others have shown that it is required for the maintenance of self renewing cells in the normal breast [1-4]. More recently, we found a group of 37 miRNAs that is differentially expressed by normal mammary stem cells. One of these miRNAs, miR-200c, regulates BMI1 and is critical for the maintenance (self-renewal) of both normal mammary stem cells and breast cancer cells (immature cancer cells) from some patients' tumors. Finally, we found that mutation of 3 downstream targets of Bmi1, p16Ink4a, p19Arf and Trp53 (triple mutant mice), allows multipotent progenitor cells to maintain themselves for long periods of time. There are 2 main goals of this grant. The first is to shed more light on the role of BMI1, its regulator (miR-200c), and its downstream targets (p16Ink4a, p19Arf and Trp53) in HSC and MMP self-renewal and maintenance. The second is to understand the impact these genes have on self-renewal in a second tissue. The results from this proposal will lead to new insights into the treatment of cancer and for the use of stem cells for regenerative medicine. We will accomplish these goals through the following specific aims. Specific Aim 1: Which population(s) of hematopoietic stem cells require Bmi1 for their maintenance? Specific Aim 2: Does miR-200c regulate self-renewal of HSCs? Specific Aim 3: To determine the cellular mechanism by which triple mutant MPPs long term engraft. Specific Aim 4: Do Trp53, p16Ink4a and p19Arf have a role in limiting self renewal in other stem cell systems? Specific populations of the stem cells will be isolated by flow cytometry and engraftment assays (to show stem cell self-renewal) for the bone marrow (long-term competitive reconstitution assay) or for mammary stem cells (mammary fat pad transplantation) will be performed. FACs analysis will be used to identify different populations of cells.